Genomic DNA fragments containing regulatory and coding sequences for the B2-subunit of the neuronal nicotinic acetylcholine receptor and transgenic animals made using these fragments or mutated fragments

ABSTRACT

Several genes encoding subunits of the neuronal nicotinic acetylcholine receptors have been cloned and regulatory elements involved in the transcription of the ∝:2 and ∝:7-subunit genes have been described. Yet, the detailed mechanisms governing the neuron-specific transcription and the spatio-temporal expression pattern of these genes remain largely uninvestigated. The β2-subunit is the most widely expressed neuronal nicotinic receptors subunit in the nervous system. We have studied the structural and regulatory properties of the 5′ sequence of this gene. A fragment of 1163 bp of upstream sequence is sufficient to drive the cell-specific transcription of a reporter gene in both transient transfection assays and in transgenic mice. Deletion analysis and site-directed mutagenesis of this promoter reveal two negative and one positive element. The positively acting sequence includes one functional E-box. One of the repressor elements is located in the transcribed region and is the NRSE/RE1 sequence already described in promoters of neuronal genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 10/843,432, filed May 12,2004, which issued as U.S. Pat. No. 7,294,463 on Nov. 13, 2007, which isa continuation of application Ser. No. 09/349,925, filed Jul. 8, 1999,which issued as U.S. Pat. No. 6,777,236 on Aug. 17, 2004, which is acontinuation of application Ser. No. 08/465,712, filed Jun. 6, 1995,which issued as U.S. Pat. No. 6,452,066 on Sep. 17, 2002, which is acontinuation of application Ser. No. 08/358,627, filed Dec. 14, 1994,which issued as U.S. Pat. No. 6,177,242 on Jan. 23, 2001, all of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to DNA and clones of β2-subunit of neuronalnicotinic acetylcholine receptor (nAChR) sequences. This invention alsorelates to genomic DNA fragments containing regulatory and codingsequences for the β2-subunit neuronal nAChR and transgenic animals madeusing these fragments or mutated fragments. The 5′ flanking sequencescontain a promoter, which confers neuron-specific expression. Thegenomic clones demonstrate the importance of the β2-subunit gene in thenicotinic system and in the pharmacological response to nicotine. Theinvention also relates to vectors containing the DNA sequences, cellstransformed with the vectors, transgenic animals carrying the sequences,and cell lines derived from these transgenic animals. In addition, theinvention describes the uses of all of the above.

References cited in this specification appear at the end by author andpublication year or by cite number.

Neuron-specific expression. Many recombinant DNA-based proceduresrequire tissue-specific expression. Unwanted or potentially harmfulside-effects of gene transfer therapies and procedures can be reducedthrough correct tissue-specific expression. Furthermore, the ability todirect the expression of certain proteins to one cell type aloneadvances the ability of scientists to map, identify or purify thesecells for important therapeutic or analytical purposes. Where the cellsof interest are neurons or a particular subset of neurons, a need forDNA sequences conferring neuron-specific or subset-specific expressionexists.

Proteins expressed throughout an organism are often utilized forspecific purposes by neurons. By expressing a particular subunit orcomponent of these proteins solely in neuronal tissue, the neurontailors the protein activity for its purposes. Finding the particular,neuron-specific subunits or components and unraveling why they areproduced only in neuronal tissue holds the key to DNA elementsconferring neuron-specific expression.

The inventors' knowledge of the biology of acetylcholine receptorsprovided an important foundation for this invention (see Changeux, TheNew Biologist, vol. 3, no. 5, pp. 413-429, May 1991). Different types ofacetylcholine receptors are found in different tissues and respond todifferent agonists. One type, the nicotinic acetylcholine receptor(nAChR), responds to nicotine. A subgroup of that type is found only inneurons and is called the neuronal nAChR.

Generally, five subunits make up an acetylcholine receptor complex. Thetype of subunits in the receptor determines the specificity to agonists.It is the expression pattern of these subunits that controls thelocalization of particular acetylcholine receptor types to certain cellgroups. The genetic mechanisms involved in the acquisition of thesespecific expression patterns could lead to an ability to controltissue-specific or even a more defined cell group-specific expression.The inventors' work indicates that defined elements in the promotersequence confer neuron specific expression for the β2-subunit.

The Pharmacological Effects of Nicotine. As noted above, nAChR respondsto the agonist nicotine. Nicotine has been implicated in many aspects ofbehavior including learning and memory (1,2). The pharmacological andbehavioral effects of nicotine involve the neuronal nAChRs. Studiesusing low doses of nicotine (23) or nicotinic agonists (16) suggest thathigh affinity nAChRs in the brain mediate the effects of nicotine onpassive avoidance behavior. Model systems where neuronal nAChR has beenaltered can therefore provide useful information on the pharmacologicaleffects of nicotine, the role of neuronal nAChR in cognitive processes,nicotine addiction, and dementias involving deficits in the nicotinicsystem.

Functional neuronal nAChRs are pentameric protein complexes containingat least one type of α-subunit and one type of α-subunit (3-5) (althoughthe α7-subunit can form functional homooligomers in vitro^(6,7)). Theβ2-subunit was selected for this study from among the 7 known α-subunitsand 3 known β-subunits (3) because of its wide expression in the brain(8-10), and the absence of expression of other β-subunits in most brainregions (10). Mutation of this subunit should therefore result insignificant deficits in the CNS nicotinic system. The inventors haveexamined the involvement of the β2-subunit in pharmacology and behavior.Gene targeting was used to mutate the β2-subunit in transgenic mice.

The inventors found that high affinity binding sites for nicotine areabsent from the brains of mice homozygous for the β2-subunit mutation,β2−/−. Further, electrophysiological recording from brain slices revealsthat thalamic neurons from these mice do not respond to nicotineapplication. Finally, behavioral tests demonstrate that nicotine nolonger augments the performance of β2−/−mice on the test of passiveavoidance, a measure of associative learning. Paradoxically, mutant miceare able to perform better than their non-mutant siblings on this task.

BRIEF SUMMARY OF THE INVENTION AND ITS UTILITY

In an aspect of this invention, we describe a 15 kb fragment of DNAcarrying regulatory and coding regions for the β2-subunit of theneuronal nAchR. We characterize the promoter of the β2-subunit gene invitro and in transgenic mice. We describe several DNA elements,including an E-box and other consensus protein-binding sequencesinvolved in the positive regulation of this gene. Moreover, we show thatthe cell-specific transcription of the β2-subunit promoter involves atleast two negative regulatory elements including one located in thetranscribed sequence.

Preferred embodiments of these aspects relate to specific promotersequences and their use in directing neuron-specific expression invarious cells and organisms. An 1163 bp sequence and an 862 bp sequenceboth confer neuron-specific expression. Other embodiments include the−245 to −95 sequence of FIG. 1, containing an essential activatorelement, and the −245 to −824 sequence of FIG. 1 containing a repressor.A repressor element composed of the NRSE/RE1 sequence is also present inthe transcribed region. Certain plasmids comprising these genomicsequences are described as well.

The promoter sequences are important for their ability to directprotein, polypeptide or peptide expression in certain defined cells. Forexample, in the transgenic mice as shown below, proteins encoding toxinsor the like can be directed to neurons to mimic the degradation of thosecells in disease states. Others will be evident from the data describedbelow.

Alternatively, the promoters can direct encoded growth factors oroncogenic, tumorigenic, or immortalizing proteins to certain neurons tomimic tumorigenesis. These cells can then be isolated and grown inculture. In another use, the promoter sequences can be operativelylinked to reporter sequences in order to identify specific neurons insitu or isolate neurons through cell sorting techniques. The isolated,purified neurons can then be used for in vitro biochemical or geneticanalysis. Reporter sequences such as LacZ and Luciferase are describedbelow.

In another aspect of this invention, the inventors provide the genomicclones for mouse β-2 subunit of the neuronal nAChR. These clones areuseful in the analysis of the mammalian nicotinic system and thepharmacology of nicotine. The inventors describe assays using transgenicmice where the genomic clones of the β2-subunit have been used to knockout the high affinity binding of nicotine.

In addition to the deletion mutants described, mutations incorporatedinto the exons or regulatory sequences for the β2-subunit will result inuseful mutant transgenic animals. These mutations can be pointmutations, deletions or insertions that result in non-efficient activityof the nAChR or even a non-active receptor. With such mutant animals,methods for determining the ability of a compound to restore or modulatethe nAChR activity or function are possible and can be devised.Modulation of function can be provided by either up-regulating ordown-regulating receptor number, activity, or other compensatingmechanisms. Also, methods to determine the ability of a compound torestore or modulate wild type behavior in the behavioral assaysdescribed or known (see 17, 22, 18, 2, 19, 21, 23, 24) can be devisedwith the mutant animals. Behavioral assays comprise, but are not limitedto, testing of memory, learning, anxiety, locomotor activity, andattention as compared to the untreated animal or patient.Pharmacological assays (see 12, 13, 14, 15, 20) to select compounds thatrestore or modulate nAChR-related activity or behavior can thus beperformed with the mutant animals provided by this invention. Dose andquantity of possible therapeutic agents will be determined bywell-established techniques. (See, for example, reference 16.)

The present model systems comprising transgenic animals or cells derivedfrom these animals can be used to analyze the role of nicotine onlearning and behavior, the pharmacology of nicotine, nicotine addiction,and disease states involving deficits in the nicotinic system. Inaddition, potential therapies for nicotine addiction or deficits in thenicotinic system can be tested with the transgenic animals or the cellsand cell lines derived from them or any cell line transfected with a DNAfragment or the complete DNA of phage β2 (CNCM accession number I-1503).These cell lines would include all those obtained directly fromhomozygous or heterozygous transgenic animals that carry or are mutatedin the β2-subunit sequences. In addition, this would include cell linescreated in culture using natural β2-subunit sequences or mutatedβ2-subunit sequences. Techniques used could be, for example, those citedin PCT WO 90/11354.

Dementias, such as Alzheimer's disease, in which the high affinitynicotine binding site are diminished suggest that the present model canbe used to screen drugs for compensation of this deficit. Accordingly,methods for screening compounds for the ability to restore or detectablyeffect activity of the neuronal nicotinic acetylcholine receptorcomprising adding the compound to an appropriate cell line orintroducing the compound into a transgenic animal can be devised.Transgenic animals and cell lines generated from this invention can beused in these methods. Such animal or cell line systems can also be usedto select compounds which could be able to restore or to modulate theactivity of the β2 gene.

The transgenic animals obtained with the β2-subunit gene sequence(wildtype or mutated fragments thereof) can be used to generate doubletransgenic animals. For this purpose the β2-subunit transgenic animalcan be mated with other transgenic animals of the same species or withnaturally occurring mutant animals of the same species. The resultingdouble transgenic animal, or cells derived from it, can be used in thesame applications as the parent β2-subunit transgenic animal.

Both the promoter sequences and the genomic clones can be used to assayfor the presence or absence of regulator proteins. The gel shift assaysbelow exemplify such a use. The sequences or clones can also be used asprobes by incorporating or linking markers such as radionuclides,fluorescent compounds, or cross-linking proteins or compounds such asavidin-biotin. These probes can be used to identify or assay proteins,nucleic acids or other compounds involved in neuron action or theacetylcholine receptor system.

Known methods to mutate or modify nucleic acid sequences can be used inconjunction with this invention to generate useful β2 mutant animals,cell lines, or sequences. Such methods include, but are not limited to,point mutations, site-directed mutagenesis, deletion mutations,insertion mutations, mutations obtainable from homologous recombination,and mutations obtainable from chemical or radiation treatment of DNA orcells bearing the DNA. DNA sequencing is used to determine the mutationgenerated if desired or necessary. The mutant animals, cell lines orsequences are then used in the DNA sequences, systems, assays, methodsor processes the inventors describe. The mutated DNA will, bydefinition, be different, or not identical to the genomic DNA. Mutantanimals are also created by mating a first transgenic animal containingthe sequences described here or made available by this invention, with asecond animal. The second animal can contain DNA that differs from theDNA contained in the first animal. In such a way, various lines ofmutant animals can be created.

Furthermore, recombinant DNA techniques are available to mutate the DNAsequences described here, as above, link these DNA sequences toexpression vectors, and express the β2-subunit protein or mutant derivedfrom the β2-subunit sequences. The β2-subunit or mutant can thus beanalyzed for biochemical or behavioral activity. In such a way, mutatedDNA sequences can be generated that prevent the expression of anefficient nAchR.

Alternatively, the promoter sequences described can be used inexpression vectors or systems to drive expression of other proteins.Obtainable DNA sequence can thus be linked to the promoter or regulatorysequences the inventors describe in order to transcribe those DNAsequences or produce protein, polypeptide, or peptides encoded by thoseDNA sequences.

DESCRIPTION OF THE RELATED ART

Previous studies by in situ hybridization (Wada et al., 1989; Hill etal., 1993; Zoli et al., 1994) and immunohistochemistry (Hill et al.,1993) demonstrate that all of the neuronal nAchR subunits cloned to datedisplay a strict neuron-specific distribution. But different subunitsexhibit an even tighter distribution to only small subsets of neurons inthe brain. For example, the nAchR ∝2-subunit transcripts are onlydetected in the Spiriformis lateralis nucleus in the chick diencephalon(Daubas et al., 1990) or the Interpeduncularis nucleus in the rat (Wadaet al., 1988). Also the β3, β4 and ∝3-subunit transcripts are onlydetected in a small set of structures in vertebrate brain (references inZoli et al., 1994).

The nAchR, ∝4, ∝5, ∝7, and β2-subunit gene transcripts, in comparison,show a much wider distribution. (Wada et al., 1989; references in Zoliet al., 1994). For example, the β2-subunit transcripts are found in themajority of neurons in the CNS and in all the peripheral neurons thatexpress the nAchR (Role, 1992; Hill et al., 1993).

As a consequence of the differential expression of these subunits, awide diversity of nAchR species occurs in vertebrates. Each species hasa defined pattern of expression involving diverse categories or groupsof neurons. For example, the neurons from medial Habenula interconnectwith those from the Interpeduncularis nucleus and yet each expressdistinct sets of nAchR subunits (see Role, 1992 for review) exhibitingdifferent physiological and pharmacological profiles (Mulle et al.,1991).

Only limited information is available, to date, about the geneticmechanisms that account for regulation of nAchR gene transcription inneurons. Previous work on the promoter of the chick ˜7 subunit geneanalyzed in vitro failed to characterize the DNA elements responsiblefor transcriptional regulation (Matter-Sadzinski et al., 1992). Inanother study, the promoter of the ∝2-subunit gene was partiallycharacterized and a silencer described and sequenced (Bessis et al.,1993, see also Daubas et al. 1993).

Certain evidence leads to the study of the β2-subunit in particular. Itis expressed in the majority of the neurons in the brain (Hill et al.,1993). Also, the timing of the appearance of the β2-transcripts closelyparallels that of neuronal differentiation (Zoli et al., 1994). We thusdecided to study the genetic mechanisms that regulate its transcription.

BRIEF DESCRIPTION OF THE INVENTION

Gene Structure

We have cloned a genomic fragment containing the regulatory sequencesand sequences encoding the mouse nAchR β2-subunit gene. The inventorshave found that at least part of the regulatory region is conservedamong different mammalian species. Particularly, the region between +16to +38 bp corresponding to the NRSE/RE1 as described in FIG. 1. UsingRNase protection and amplification of primer extension products, wefound one main and three minor transcription start sites (FIG. 1). Theprimer extension experiments were performed using two different reversetranscriptases, with different batches of mRNA and with differentprimers. These PCR based techniques allowed us to amplify and subclonethe same fragments corresponding to transcription start sites ratherthan reverse transcriptase stops. The transcription start sites that wehave characterized are located downstream from the position of thelongest rat (Deneris et al., 1988) and human (Anand and Lindstrom, 1990)β2 cDNA 5′ end (see FIG. 1). This implies that in human and rat, anothertranscription start site is used. Such a discrepancy between species hasalready been demonstrated for the ε-subunit of the muscle nAchR (Dürr etal., 1994, see also Dong et al., 1993; Toussaint et al., 1994). Incontrast with the α2 subunit gene (Bessis et al., 1993), no upstreamexon could be detected.

Structural analysis of a 1.2 kbp flanking region disclosed manyconsensus motifs for nuclear protein binding including an Sp1 site andan E-box. Approximately 90 bp of the undeleted 1.2 kb promoter aretranscribed and this region contains a NRSE/RE1 sequence (Kraner et al.,1992; Mori et al., 1992). Regulatory elements have already beendescribed downstream of the transcription start site in differentsystems such as the Polyomavirus (Bourachot et al., 1989) or the fosgene (Lamb et al., 1990).

The promoter region is located between the Eco47III located in exon 1(see FIG. 1) (SEQ ID NO: 22) and the BamHI site 4.5 kb upstream. Onepreferred embodiment is the 1163 bp sequence described in FIG. 1 betweenthe EcoRI and Eco47III sites. Regulatory sequences may be located in the2 kb downstream from the Eco47III site. The regulatory elements from thenAchR β2-subunit sequences can be used to direct the neuron specificexpression of a nucleotide sequence encoding a protein, polypeptide orpeptide linked to them. Said protein, polypeptide, or peptide can betoxins, trophic factors, neuropeptides, tumorigenic, oncogenic, orimmortalizing proteins, or any other protein that can change thefunction of the neuron.

A 1163 bp Promoter Achieves Cell-Specific Transcription.

The 1163 bp promoter contains regulatory sequences for bothtissue-specific and temporal specific transcription of the β2-subunitgene. Transient transfection experiments showed that the 1163 bpfragment contains sufficient information to confer cell-specificexpression of the nAchR β2-subunit gene. We showed that the samepromoter directs a strict cell-specific transcription of theβ-galactosidase (β-gal) reporter gene. Moreover, the transgenicconstruct appears to be activated with the same timing as the endogenousβ2-subunit gene during the development of the early embryonic nervoussystem (Zoli et al., 1994). At later stages of development, most of theperipheral β2 expressing neurons are still labelled (FIGS. 4C, D).

The promoter sequence was tested in transgenic mice by generating twolines (13 and 26) expressing β-gal under the control of the 32-subunitpromoter. In CNS, the pattern of β-galactosidase expression is differentbetween the two lines. Only a subset of the cells that normally expressβ2 express the transgene. This type of discrepancy between theexpression of the transgene and the endogenous gene has already beendescribed for the dopamine β-hydroxylase gene promoter (Mercer et al.,1991; Hoyle et al., 1994) or for the GAP-43 gene (Vanselow et al.,1994). Unexpected expression has been observed in transgenic line 13 inthe genital tubercule and in skin muscles. This expression is likely tobe due to the integration site of the transgene as these tissues are notstained in line 26. To our knowledge, most of the neuronal promotersstudied by transgenesis display ectopic expression in a certain smallpercentage of transgenic lines (Forss-Petter et al., 1990; Kaneda etal., 1991; Banerjee et al., 1992; Hoesche et al., 1993; Logan et al.,1993, Vanselow et al., 1994). However, techniques in the art afford theconstruction of lines where the expression pattern of the transgeneclosely mirrors or duplicates that of the original gene. See referencesfor further details showing the success of the transgenesis procedure.

By comparing the β-gal positive cell distribution with those of otherknown neuronal markers, it becomes apparent that a similarity existswith the distribution of choline acetyltransferase, TrkA (the highaffinity nerve growth factor receptor) and p⁷⁵ (the low affinity nervegrowth factor receptor) expressing cells (Yan and Johnson, 1988: Pioroand Cuello, 1990a, b; Ringstedt et al., 1993). In particular, indeveloping rats, p⁷⁵ is expressed in almost all the peripheral gangliaand central nuclei (with the exception of the zona incerta andhypothalamic nuclei), which express the transgene (Yan and Johnson,1988). It is also interesting to note that p⁷⁵ expression (like theexpression of the β2-promoter transgene) is transient in many peripheralganglia and brain nuclei, decreasing to undetectable levels at perinatalor early postnatal ages. It is therefore possible that the β2-subunitpromoter contains an element controlled by the activation of p⁷⁵, orthat both the β2 transgene and p⁷⁵ gene are controlled by a commonregulator.

In conclusion, although the promoter seems to lack some regulatoryelements active in the brain, the existing regulatory elements aresufficient to allow a cell- and development-specific expression ofβ-galactosidase in the PNS, in the spinal cord, and in several brainstructures. The promoter can also be used in assays to identifyregulator proteins in neuronal tissue.

DNA Regulatory Elements.

To further characterize the DNA elements involved in the transcriptionof the β2 subunit gene, we deleted or mutated the 1163 bp promoter andanalyzed the resulting constructs by transient transfection. A repressorelement present in the distal 5′ end region is active in fibroblasts butnot in neuroblastomas. This element thus accounts, at least in part, forthe neuron-specific expression of the β2-subunit gene. Further analysisof the promoter shows that deleting 589 bp increases the activity inneuroblastomas, but not in fibroblasts (FIG. 6, compare 862E and283E-Luci).

An NRSE/RE1 element is located at the 3′ extremity of the promoter. Thiselement has already been shown to restrict the activity of promoters inneuronal cells (Kraner et al., 1992; Mori et al., 1992; Li et al.,1993). In the 1163 bp promoter of the β2-subunit gene, point mutation ofthis sequence leads to a ˜100 fold increase of the transcriptionalactivity in fibroblasts implying that this sequence is involved in theneuron-specific expression of the β2-subunit gene. Moreover, sequencecomparison shows that this sequence is highly conserved in rat and humanβ2-subunit cDNAs (Deneris et al., 1988; Anand and Lindstrom, 1990) aswell as in several promoters of genes expressed in the nervous system,such as the middle-weight neurofilament gene, the CAM-L1 gene, theCalbinbin gene, or the cerebellar Ca-binding protein gene (see Table1B).

Deletion experiments described in FIG. 6 show that an essentialactivator element is present between nucleotides −245 and −95. An Sp1binding site and an E-box could be detected in this region. Sp1 sitesare ubiquitous factors, whereas E-boxes have been involved in severalgenetic regulatory mechanisms in muscle (see Bessereau et al., 1994 forthe nAchR ∝1-subunit) as well as in neurons (Guillemot et al., 1993).Dyad elements have also been reported in some neuronal promoters, suchas those of the Tyrosine hydroxylase gene (Yoon and Chikaraishi, 1994),the SCG1O gene (Mori et al., 1990), the GAP43 gene (Nedivi et al.,1992), or in the flanking region of the N-CAM gene (Chen et al., 1990).Results shown in Table 1[[A]] demonstrate that in neuroblastomas, the1163 bp promoter mutated in the E-box/Dyad is significantly less activethan the wild type promoter. Moreover, a gel shift assay (FIG. 7)further demonstrates that the E-box/Dyad is able to bind specificcomplexes. This suggests that the E-Box/Dyad is responsible for at leastpart of the activation of β2-subunit gene transcription. However,transactivation experiments of heterologous promoters suggest that theE-box cooperates with the Sp1 site located 27 bp upstream to positivelyactivate transcription. This type of cooperation between an E-Box and anSp1 binding site has already been demonstrated for the regulation of themuscle nAchR ∝1-subunit transcription (Bessereau et al., 1993).

In conclusion, we have shown that the β2-subunit gene is primarilyregulated by negatively acting elements and by one positive element thatcomprises an E-box. This double regulation seems to be a general featureshared by several neuronal genes (Mandel and Mckinnon, 1993) and allowsfine tuning of the transcription of neuronal genes. Moreover, ourtransgenic studies show that the 1163 bp promoter confers a tightneuron-specific expression, but lacks some developmental or CNS-specificregulatory elements.

DESCRIPTION OF THE FIGURES

FIG. 1: Nucleotide sequence of the region surrounding the initiator ATGof the β2-subunit gene.

The four vertical arrowheads show the four extremities found usingRACE-PCR and SLIC, corresponding to the transcription start sites. Thevertical arrows indicate the position corresponding to the 5′ end of thelongest rat (r) and human (h) β2-subunit cDNA clones (Deneris et al.,1988). The endpoints of the deletions used in the experiments describedin FIG. 3 are indicated above the sequence. Nucleotides located in theintron are typed in lower cases.

FIG. 2: Mapping of the 5′ end of the β2-subunit mRNA.

A. RNase protection experiments. Total RNA from DBA2 mouse brain (5 and15 μg, lane 2 and 3 respectively) and yeast tRNA (15 μg, lane 1) werehybridized to a ³²P-labeled RNA probe containing 158 nucleotides ofintron 1, and 789 nucleotides of upstream sequences (−634/+155). Thesize of the protected bands were estimated according to the lowermobility in acrylamide of RNA as compared to DNA (Ausubel et al., 1994)and by comparison with the sequence of M13mp18 primed with the universalprimer. The arrow on the left part of the gel points to the majorprotected band.

B. Identification of the transcription start site using SLIC. The lowerpart of the Figure shows the strategy and describes the oligonucleotidesused for the SLIC or the RACE-PCR. In the SLIC experiment, a primerextension was performed using oligonucleotide pEx3. The first strand ofthe cDNA was subsequently ligated to oligonucleotides A5′, and theresulting fragment was amplified using oligonucleotides A5′-1/p0 thenA5′-2/p1. The amplified fragment was then loaded onto a 1.2% agarosegel. The gel was blotted and hybridized to oligonucleotide p2. Lane 1: 5μg of total DBA2 mouse brain RNA. Lane 2-3: controls respectivelywithout reverse transcriptase and without RNA. Minus: the T4 RNAPolymerase was omitted. Same result was obtained using RACE-PCR.

FIG. 3: Cell-specific expression of the β2-subunit promoter in vitro.

The luciferase activity of the plasmids were normalized to the activityof the promoterless plasmid (KS-Luci, described in Materials andMethods). RACE-PCR on mRNA extracted from SK-N-Be transfected withEE1.2-Luci, using luciferase oligonucleotides (described in Material andMethods) showed that the amplified fragment had the expected size forthe correct transcription initiation site.

FIG. 4: Cell-specific expression of the β2-subunit promoter intransgenic mice.

A. Whole mount coloration of E13 embryos. The arrowheads point toectopic expression in skin muscles. B. Detection of the β3-galactosidaseactivity in a parasagittal section of an E13 embryo at the lumbo-sacrallevel. Arrowheads indicate labelling in the ventral and dorsal horn ofthe spinal cord. C. Detection of the β2-subunit transcripts in anadjacent section of the same embryo. dr: dorsal root ganglion; t:tectum; og: orthosympathetic ganglionic chain; tr: trigeminal ganglion.

FIG. 5: Expression of β3-galactosidase in transgenic mice.

A. staining of the retina(re) and the trigeminal ganglia (tr) (E14.5).

B. staining of cardiac parasympathetic ganglionic neurons (pg) (E14.5).C. transverse section of the spinal cord (P1). dr: dorsal root ganglion,og: orthosympathetic ganglion. D. Ventral view of the spinal cord (P1).The smaller arrows indicate neurons that have not been identified.

FIG. 6: Expression of the Luciferase fusion genes containing 5′ enddeletions of the β-subunit promoter.

Plasmids are called nnnE-Luci, where nnn is the size in nucleotides ofthe insertion, and E is the 5′ end restriction site (Eco47III). Thearrow indicates the transcription start site. The activities ofEE1.2-Luci are from FIG. 3.

FIG. 7: Gel shift experiment. Autoradiogram of the mobility shiftexperiment. The probe used was a ³²P labelled double stranded E-Doligonucleotide. This oligonucleotide carries only the E-Box/Dyadelement, whereas the oligonucleotide S-E carries the Sp1 binding site aswell as the E-Box/Dyad element. The competitor oligonucleotides wereused in 10- and 100-fold molar excess, except for S-E that was used onlyin 100-fold molar excess.

FIG. 8: Disruption of the gene encoding the β2-subunit of the neuronalnAChR. a-i, Normal genomic structure of the mouse β2-subunit gene.Portion of exon one removed by the recombination event is shaded inlight grey. ATG—initiator methionine. Boxes represent exons I-IV. a-ii,Targeting replacement vector used to disrupt the endogenous β2-subunitgene. Initiator methionine and the rest of the first exon were replacedwith the coding region of NLS-lacZ and the MCI neo^(R) expressioncassette²⁵. The construct was able to direct lacZ expression afterstable transfection of PC12 cells (not shown), but lacZ expression wasnever detected in recombinant animals, despite the lack of obviousrecombination in the lacZ DNA. Diphtheria toxin-A gene (DTA)²⁶ was usedto select against random integration. a-iii, Structure of the mutatedβ2-gene. Restriction sites: H, HindIII; R, EcoRI; E, Eco47III; P, PstI.Black arrows, primers used to detect recombination events in embryonicstem (ES) cells. Grey arrows, primers used to detect the wildtype ormutated β2 genes. b, PCR analysis of tail DNA from a +/+, +/− and a −/−mouse. c, Southern blot analysis of tail DNA restricted with HindIIIfrom the same mice analyzed in panel b. d, Western blot analysis oftotal brain protein using a monoclonal antibody raised against theβ2-subunit.

METHODS: a, The β2-targeting vector was constructed by inserting amultiple cloning site (MCS) into the MCI neo cassette (GTC GAC GGT ACCGCC CGG GCA GGC CTG CTA GCT TAA TTA AGC GGC CGC CTC GAG GGG CCC ATG CATGGA TCC) (SEQ ID NO: 30). A 4.1 kB EcoRI-Eco47III β2-genomic fragment 5′to the ATG and a 1.5 kB PstI β2-genomic fragment starting within thefirst intron of the β2-gene were cloned into the MCS. HM1^(27,28)embryonic stem cells (5×10⁷) were transfected with the linearizedtargeting vector by electroporation as described²⁵. Twenty-foursurviving G418-resistant clones were screened by PCR (β2-primer—GCC CAGACA TAG GTC ACA TGA TGG T (SEQ ID NO: 31); neo-primer—GTT TAT TGC AGCTTA TAA TGG TTA CA (SEQ ID NO: 32)). Four were positive and were laterconfirmed by Southern blot analysis. Clones were injected into3.5-day-old blastocysts from non-agouti, C57BL/6 mice and planted inreceptive females. All resulting male chimaeric mice were mated to F1,C57BL/6xDBA/2 non-agouti females. Of 15 chimaeras, one showed germ-linetransmission. β2+/− heterozygotes were mated and offspring wereevaluated by PCR analysis (panel b). b, PCR was 35 cycles of 94°/1 min,65°/2 min and 72°/1 min. c, Southern blotting was performed asdescribed²⁹. The 1.5 kB PstI genomic fragment used for the targetingconstruct was labelled by random priming. d, Western blotting wasperformed as described²⁹ using monoclonal antibody 270¹¹.

FIG. 9: Mapping of the neuronal nAChR in mouse brain using in situhybridization and tritiated nicotine binding. A, In situ hybridizationusing antisense oligonucleotide probes based on the sequence of thecDNAs encoding the β2-, α4- and β4-subunits of the nAChR to detect theirrespective mRNAs in serial sections from the brains of 32+/+, +/− and−/− mice. Midthalamic sections are shown. White arrows indicate the MHblabelled by the β4-antisense oligonucleotide. B, Receptorautoradiography using tritiated nicotine revealing high affinity bindingsites in the brains of wildtype, heterozygous and β2-mutant mice.Representative sections at the level of the striatum, thalamus andtectum are shown.

METHODS, A, In situ hybridization was performed as follows:

In situ hybridization procedure. Frozen tissues were cut at the cryostat[14 μm thick sections), thaw mounted on poly-1-lysine coated slides andstored at −80 C for 1-3 days. The procedure was carried out according toYoung et al. (1986). Briefly, sections were fixed with 4%paraformaldehyde for 5 min. at room temperature, washed in phosphatebuffered saline (PBS) and then acetylated and delipidated in ethanol andchloroform (5 min.). They were prehybridized for 2-4 h at 37 C underparafilm coverslips. The composition of the prehybridization andhybridization mixtures was 50% formamide, 0.6M NaCl, 0.1Mdithiothreitol, 10% dextran sulfate, 1 mM ethylenediaminetetraaceticacid (EDTA), 1×Denhardt's solution (50×=1% boyine serum albumin/1%Ficoll/1% polyvinylpyrrodlidone), 0.1 mg/ml polyA (Boehringer), 0.5mg/ml yeast RNA (Sigma), 0.05 mg/ml herring sperm DNA (Promega) in 0.02MTris-HCl, pH 7.5. Probes were applied at a concentration of 2000-3000Bcq/30 μl section (corresponding to around 15 fmol/section). Afterremoval of coverslips and initial rinse in 2× standard saline citrate(SSC) solution (3M NaCl/0.3M sodium citrate) at room temperature (twotime for 5 min.), sections were washed four times for 15 mm in 2×SSC/50%formamide at 42° C. and, then, two times for 30 min in 1×SSC at roomtemperature. 1 mM dithiothreitol was added to all washing solutions.After rinsing in ice-cold distilled water and drying, they were exposedfor 10-20 days to Hyperfilm βmax (Amersham) and then to a photographicemulsion (NTB2, Kodak) for 1-2 months.

Analysis of histological preparations. The analysis of the labellingpattern for the different mRNAs was carried out both on film andemulsion autoradiograms. Identification of anatomical structures wascarried out after counterstaining of the serial sections of the entireembryos with toluidine blue. Definition of anatomical areas in the brainand recognition of peripheral nervous system (PNS) structures was basedon different atlases, including The Rat Brain in Stereotaxic Coordinates(Paxinos and Watson, 1986), the Atlas of Developing Rat Brain (Paxinoset al. 1991), the Atlas of Mouse Development (Kaufman, 1992), and theAtlas of the Prenatal Mouse Brain (Schambra et al., 1992). For cranialnerve ganglia development, the plates and descriptions from Altman andBayer (1982) were consulted. In order to confirm the identification ofsome central and peripheral structures (e.g., cranial nerve motornuclei, autonomic motor ganglia) in situ hybridization for cholineacetyltransferase was performed on some sections.

A score from 1+ (low intensity) to 3+ (high intensity) was assigned tothe labelling of the anatomical structures based on the subjectiveevaluation of two experimenters. Background labelling was considered thedensity of grains in nonneural tissues high cellularity (such as theliver and muscles) or with high density of extracellular matrix (such ascartilage) or the density of labelling over neural structures afterdisplacement with 20× cold probe. In the absence of grain counting atthe cellular level, the scores must be regarded with caution. Forinstance, decreases in labelling intensity of a developing structure maybe due to dispersion of positive cells in the structure caused bymultiplication of negative cells or formation of neuronal processes.Though the oligonucleotides had the same length and they were labelledaccording to the same protocol, no attempt to compare the signalintensity or different transcripts was made. Unless specified otherwise,the labeling shown in the pictures has been obtained by usingoligonucleotides no. 31 (∝3), 47 (∝4), 51 (†2), and 62 (∝4) (see Table 1for oligonucleotide characteristics).

Specificity controls. For each mRNA, two to four oligonucleotides wereselected in unique parts of the sequence (e.g., the putative cytoplasmicloop between M3 and M4 for nAChR subunits). An initial assessment of thespecificity was performed by searching for possible homology with otherknown sequences in Genbank/EMBL. As histological tests for specificitywere considered the following: 1. Two or more oligonucleotide probes foreach mRNA gave the same hybridization pattern (FIG. 1). 2. The patternof labelling in central structures in the adult rat was in agreementwith that observed by other authors (Wada et al., 1989; Dineley-Millerand Patrick, 1992). 3. Given that most oligonucleotides used were45-mers with similar GC content (Table 1), each oligonucleotide probeconstituted a control for the specificity of the others. 4. The additionto the hybridization mixture of a 20-fold excess of cold probe produceda complete disappearance of the labelling (FIG. 2).

The oligonucleotide probes used fulfilled all these criteria, with theexception of the four probes against ∝3 mRNA, which did not satisfycriterion 2. Previous studies based on cRNA probes showed a relativelywidespread distribution of this subunit mRNA in adult rats, notably highlevels in the cerebral cortex layer IV, entorhinal cortex layer II,anterior and ventral thalamic nuclei, medial and lateral geniculatenuclei, medial habenula, posterior hypothalamus and supramammillarynuclei, pineal gland, motor nuclei of the V and VII nerves, locuscoeruleus, nucleus ambiguus, and area postrema (Wada et al. 1989). Atvariance with these observations, in adult rats we could detect highlevels of ˜3 mRNA signal only in the medial habenula, intermediate inthe pineal gland, area postrema, motor nucleus of the V nerve andcerebellum, low in a few thalamic nuclei and locus coeruleus. Part ofthe discrepancy may be ascribed to a lower sensitivity ofoligonucleotide probes versus riboprobes. However, considering thedifficulty of carrying out specificity controls for cRNA probes,especially when hydrolysis of the probe is performed in the histologicalprocedure (Wada et al., 1989), it is possible that some labellingpreviously attributed to ∝3 mRNA actually derives from hybridization toother (nAChR-related) RNA sequences. Oligonucleotides: β2 (SEQ ID NO:1): 5′-TCG CAT GTG GTC CGC AAT GAA GCG TAC GCC ATC CAC TGC TTC CCG-3′;α4 (SEQ ID NO: 2): 5′-CCT TCT CAA CCT CTG ATG TCT TCA AGT CAG GGA CCTCAA GGG GGG-3′; α4 (SEQ ID NO: 3):5′-ACC AGG CTG ACT TCA AGA CCG GGA CGCTTC ATG AAG AGG AAG GTG-3′. B, ³H-nicotine binding was performed asdescribed by Clarke et al³⁰. Fourteen μm coronal sections were incubatedat room temperature for 30 min. in 50 mM Tris pH 7.4/8 mM CaCl₂/4 nM³H-L-nicotine. Nonspecific binding was evaluated in the presence of 10μM L-nicotine bitartrate. Following incubation, sections were rinsed 2×2min. in ice cold PBS and briefly rinsed in ice cold water. Slides wereexposed for 60 days to Hyperfilm ³H.

FIG. 10: Patch clamp recording of nicotine evoked currents in the MHband anterior thalamus of β2+/+ and −/− mice. A, Representativerecordings from cells in the MHb and the anterior thalamus of wildtypeand β2−/− mice. The off-rate of the agonist is significantly greater inthe MHb than in the anterior thalamus, resulting in a different kineticsof response in the two structures. The response to nicotinic agonists ofthe MHb is maintained in 32−/− animals, while the response to nicotinicagonists of the anterior thalamus is completely abolished in 32−/− mice.B, table of responses to nicotinic agonists in various nuclei of 32+/+and −/− mice. METHODS, Coronal slices were obtained from the thalamus of8-12 day old mice using a Dosaka slicer in ice cold ACSF medium (125 mMNaCl/26 mM NaHCO₃/25 mM Glucose/1.25 mM NaH₂PO₄/2.5 mM KCl 2.5/2 mMCaCl₂/1 mM MgCl₂ pH 7.3). Slices were maintained in the same medium for1-8 hours. Cells in slices were visualized through a Zeiss microscope.Whole cell recordings were obtained with 2-4 MOhm hard-glass pipettescontaining 150 mM CsCl/10 mM EGTA/10 mM HEPES/4 mM di-sodium ATP/4 mMMgCl₂ pH adjusted to 7.3 with KOH. Five to ten sec. pulses of drug wereapplied rapidly to the cell through a 50 μM diameter pipette above theslice, fed by gravity with a solution containing 150 mM NaCl/10 mMHepes/2.5 mM KCl/2 mM CaCl₂/1 mM MgCl₂. Recordings were made in thepresence of CNQX (5 μM) and of the GABA_(A) antagonist SR-95531 (10 μM).Currents were recorded with an Axopatch ID (Axon Instrument) patchamplifier, digitized on a Compaq PC and further analyzed with the PClampprogram (Axon Instrument).

FIG. 11: Performance of β2−/− mice and their wildtype siblings on thepassive avoidance test. A, response to various levels of footshock inretention test following a post-training injection of either vehicle ornicotine (10 μg/kg). Average step-through latency during the trainingtrial was 17.0+/−3.6 sec for mutant mice and 15.0+/−3.5 sec for theirnonmutant siblings. B, bar graph showing the difference in retentionlatency between wildtype and homozygous β2 mutant mice injected witheither vehicle or nicotine (10 μg/kg) at foot shock intensity of 2.00mAmp. Data are represented as means+/−S.E.M. of the following groups:wildtype+vehicle (n=27); wildtype+nicotine (n=23); β2-mutantmice+vehicle (n=17); β2-mutant mice+nicotine (n=17). Statisticalanalysis was performed using a mixed factorial analysis of variancefollowed by a-posteriori testing of simple effects. #, p<0.05, wildtypevs mutant mice following vehicle injection; *, p<0.01, nicotine vsvehicle in wildtype mice.

METHODS, Passive avoidance test was performed as described in the text,according to Nordberg and Bergh²⁰ and Faiman et al²⁰. Nicotine(bitartrate, Sigma) was freshly dissolved in PBS. IP injection of thesame volume of either nicotine or vehicle immediately followed footshockduring the training trial.

FIG. 12: Phage and plasmids containing all or part of the β2-subunitgene and the promoter. In the names of the plasmids, the numeralsindicate the size of the fragment and the letters indicate therestriction sites used to generate it.

DETAILED DESCRIPTION

The descriptions and examples below are exemplary of the embodiments andscope of this invention. The invention is not limited to the scope ofthis description. Furthermore, this description together with theaccompanying sections of this specification and the materialincorporated by reference enables the practice of all of the claimswhich follow.

The examples and embodiments that follow of course can be modified bytechniques known in the art. Variations in the nucleic acid sequencesdescribed or claimed can be produced by known methods without alteringthe effects or advantages the inventors have shown. Such variations aretherefore included within the scope of this description and invention.

Materials and Methods

Isolation of Genomic Clones.

The PCX49 plasmid (Deneris et al., 1988) containing the entire rat cDNA(kindly provided by Drs. J. Boulter and S. Heinemann, The SalkInstitute, San Diego, Calif.) was cut with EcoRI, the ˜.2.2 kb fragmentwas isolated and used as a probe to screen an EMBL3 bacteriophagelibrary of mouse DBA2 genomic DNA. One unique clone was obtainedspanning ˜15 kb of DNA upstream and ˜5 kb downstream from the firstexon. FIG. 1 shows the nucleotide sequence of 1.2 kb upstream from theinitiator ATG.

Hybridization conditions can be modified by known techniques²⁹ todetermine stringent conditions for this probe. Changes in thehybridization conditions such as temperature (from about 45° C. to about65° C.) and SSC buffer concentration (from about 0.1×SSC to about6×SSC), as well as changes in the temperature of and the buffer for thewashing conditions can be made to develop sufficiently stringentconditions that allow hybridization to the β2-subunit sequences. Otherrelated sequences can thus be isolated from other libraries based onthis hybridization procedure. Human sequences will be isolated by usinghybridization conditions such as 45° C. and 6×SSC.

Three deposits were made on Dec. 13, 1994 at the Collection Nationale deCultures de Microorganismes (CNCM), Institut Pasteur, 25 Rue du DocteurRoux, 75724 PARIS CEDEX 15, France. A phage, λβ2 nAchR, is depositedunder the accession number I-1503. This phage contains 15-20 kb ofgenomic DNA including the promoter sequences and the coding sequencesfor all of the exons of the murine 32-subunit of neuronal nAchR. Two E.coli cultures bearing plasmids have also been deposited. Plasmid pSA9 inE. coli DH5α has accession number I-1501 and contains 9 kb of murinegenomic DNA including the regulatory sequences and regions coding forexons 1, 2 and 3 of the β2-subunit. Plasmid pEA5 in E. coli DH5α hasaccession number I-1502 and contains 5 kb of murine genomic DNAincluding a region of about 1.2 kb upstream of the Eco47-III site and aregion coding for exons 1 to 5 of the β2-subunit. The inventors intendto deposit the nucleotide sequence data reported here in the EMBL,GenBank and DDBJ Nucleotide Sequence Databases under the accessionnumber: X82655.

Mapping of the Transcription Initiation Site.

For the mRNA mapping, we used different batches of total RNA extractedfrom DBA2 embryos at stage E13 or E15. The RNA samples were firstdigested with DNase I to avoid DNA contamination.

RNase protection. An XbaI/PstI fragment containing part of intron 1 wasinserted into Bluescript SK (Stratagene). The plasmid was thenlinearized by BgIII, and an RNA probe was synthesized using the T7promoter. The protection experiments were then performed as described inAusubel et al. (1994).

RACE-PCR (Frohman et al., 1988). The mRNA was hybridized 5 minutes at80° C. with 10 pmol of primer. The synthesis of the cDNA was performedusing 400u MMLV (Gibco) for 45 minutes at 37° C. in the bufferrecommended by the supplier. After a phenol/chloroform extraction, thecDNA was ethanol precipitated. The terminal transferase reaction wasperformed in 0.2 M potassium cacodylate; 25 mM Tris-HCl pH 6.6; 25 mg/mlBSA; 1.5 mMCoCl₂; 50 nM dATP and 50u Terminal transferase (Boehringer)for 30 minutes at 37° C. After phenol/chloroform extraction and ethanolprecipitation, one tenth of the terminal transferase reaction wasamplified using Promega's Taq DNA polymerase (30 cycles, 1 minute at:94° C.; 55° C.; 72° C.). The amplified fragment was then loaded on anagarose gel. The gel was blotted and hybridized to oligonucleotide p2.We used pEx2 as a primer for cDNA synthesis, and p0/BEpT for PCR to mapmRNA from brain. OLUCI3 (synthesis of cDNA) and OLUCI2/BEpT (PCR) wereused to map mRNA from transfected cells. SLIC (Dumas Milnes Edwards etal., 1991). The cDNA was first synthesized from 5 μg total RNA usingpEx3 (6 pmol) as a primer in 50 mM Tris-HCl pH 8.3; 8 mM KCl; 1.6 mMMgCl₂; 5 mM spermidine; 0.5 mM dNTP; 1 u/μl RNasin; 0.1 mg/ml BSA; 70 mMβ-mercaptoethanol; 80u AMV reverse transcriptase (Promega) at 42° for 45minutes. The RNA was subsequently degraded in NaOH. The first strand ofthe cDNA was then ligated with the oligonucleotide A5′. The resultingsingle stranded cDNA was then submitted to two rounds of PCRamplification with oligonucleotides A5′-1/p0 and A5′-2/pl (35 cycles 94°C. 1 minute; 60° C. 30 seconds; 72° C. 45 seconds).

The sequence of the oligonucleotides were the following:

A5′ (SEQ ID NO: 4): 5′-CTGCATCTATCTAATGCTCCTCTCGCTACCTGCTCACTCTGCGTGACATC A5′-1 (SEQ ID NO: 5): 5′-GATGTCACGCAGAGTGAGCAGGTAG A5′-2 (SEQ ID NO:6): 5′-AGAGTGAGCAGGTAGCGAGAGGAG p0 (SEQ ID NO: 7):5′-CCAAAGCTGAACAGCAGCGCCATAG p1 (SEQ ID NO: 8):5′-AGCAGCGCCATAGAGTTGGAGCACC p2 (SEQ ID NO: 9):5′-AGGCGGCTGCGCGGCTTCAGCACCACGGAC pEx2 (SEQ ID NO: 10):5′-GCCGCTCCTCTGTGTCAGTACCCAAAACCC pEx3 (SEQ ID NO: 11):5′-ACATTGGTGGTCATGATCTG BEpT (SEQ ID NO: 12):5-GCGGGATCCGAATTC(T)₂₁ A/C/G OLUCI3 (SEQ ID NO: 13):5′-CGAAGTATTCCGCGTACGTGATG OLUCI2 (SEQ ID NO: 14):5′-ACCAGGGCGTATCTCTTCATAGC

Construction of Plasmids.

KS-Luci: The HindIII/KpnI restriction fragment of the pSVOAL plasmid (deWet et al., 1987) was subcloned in the corresponding site of BluescriptKS. The most 5′ EcoRI/BsmI (45 bp) fragment of the Luciferase gene wasthen deleted according to (de Wet et al., 1987) and replaced by a Sal Isite. The 342 bp PvuII/HindIII restriction fragment of SV40 containingthe polyadenylation sites was subsequently subcloned into the EagI sitesusing adaptors.

EE1.2-Luci: The 1.2 kbp EcoRI/Eco47II fragment of the λβ2 phage wasinserted in the EagI/SalI sites of KS-Luci using adaptors. The 5′ enddeletions of the promoter were obtained using Bal3.1 exonuclease as inCurrent Protocols in Molecular Biology (Ausubel, et al., 1994).

The mutations were introduced using the Sculptor kit (Amersham). In theNRSE/RE1 sequence, the mutated sequence was: +24 ACCACTTACA (SEQ ID NO:15) instead of ACCACGGACA (SEQ ID NO: 16), as this mutation was shown toreduce the activity of the NRSE element (Mori et al., 1992). In theE-box sequence, the mutated sequence was: −120 TCCTCAGG (SEQ ID NO: 17)instead of TCCACTTG (SEQ ID NO: 18). FIG. 7 shows that a nuclear proteinis able to bind to the wild type sequence, but not to the mutatedsequence.

Transfection of Cells.

Neuroblastomas N1E115, human SK-N-Be, HeLa and 3T6 fibroblasts, 293Human kidney cells and SVLT striatal cells (Evrard et al., 1990) weregrown in DMEM+10% FCS supplemented with 1% glutamine and 1%streptomycin. PC12 cells were grown in DMEM+10% HS+5% FCS supplementedwith 1% glutamine and 1% streptomycin.

Cells were plated at 10⁵ to 4×10⁵ cells/60 mm² plates. The next daycells were transfected in 750 μl of DMEM+2% Penicillin/Streptomycin for5 to 12 hours with 1 μg DNA mixed with 2.5 μl of Transfectam(IBF/Sepracor) in 150 mM NaCl. The Luciferase activity was measured 48hours later. DNA was prepared using Qiagen or Wizard prep (Promega)kits. When plasmid activities were compared, all plasmids were preparedthe same day. At least two different DNA preparations were tested foreach plasmid. All transfections were done in duplicate and repeated atleast three times. Production of Transgenic Mice.

The luciferase gene from EE1.2-Luci was excised and replaced by thenlsLacZ gene (Kalderon et al., 1984). The β2-promoter/nlsLacZ fragmentwas electroeluted from a TAE agarose gel then further purified byethanol precipitation, and finally resuspended in Tris-HCl 10 mM pH 7.5;EDTA 0.1 mM. The DNA solution (3 ng/ml) was injected into fertilizedoocytes of C57BL6xSJL hybrids. Staining of tissues was performed asdescribed in Mercer et al., 1991. See also the methods under FIG. 8.

Gel Shift Assay

Oligonucleotides were labeled either with γ[³²P]ATP and T4polynucleotide kinase, or with ∝[³²P]CTP and Klenow enzyme as in CurrentProtocols in Molecular Biology. Nuclear extracts were prepared from ≅10⁷cells as described (Bessis et al., 1993). For binding, 1 nmol of labeledoligonucleotide was mixed with 0.5 μg of protein extract in 10 mM HepespH 8, 10% glycerol, 0.1 mM EDTA, 0.1 M NaCl, 2 mM DTT, 0.1 mg/ml BSA, 4mM MgCl₂, 4 mM spermidine, 1 mM PMSF, 1 μg polydldC in 20 μl. Thereaction was incubated for 10 minutes on ice. The DNA-protein complexeswere then analyzed on a 7% polyacrylamide gel.

The oligonucleotides used in this experiments were double stranded withthe following sequences (the nucleotides in bold are changed between themutated and the wild type oligonucleotides):

E-D (SEQ ID NO: 19): 5′-TCCTCCCCTAGTAGTTCCACTTGTGTTCCCTAG Mut-E (SEQ IDNO: 20): 5′-CCTCCCCTAGTAGTTCCTCAGGTGTTCCCTAGA S-E (SEQ ID NO: 21):5′-CTAGCTCCGGGGCGGAGACTCCTCCCCTAGTAGTTCCACTTGTGTTC CCTAG

Results

Characterization of the 5′ Flanking Sequences of the Gene Encoding theβ2-Subunit

A λ phage containing the gene encoding the β2-subunit was cloned and aregion surrounding the initiator ATG was sequenced (FIG. 1). Thetranscription initiation site was first mapped by RNase protection (FIG.2A). This method allowed us to detect at least three initiation sites.However, minor additional start sites might not have been detected inthese experiments. The size of the main protected band was estimated atabout 150 nucleotides. To confirm and locate the initiation sites moreprecisely, we performed both RACE-PCR (Rapid Amplification of cDNA Ends;Frohman et al., 1988) and SLIC (Single Strand Ligation of cDNA; DumasMilnes Edwards et al., 1991) which consist in the amplification of theprimer extension product (FIG. 2B). Both techniques allowed us tosubclone and sequence the same fragments corresponding to the fourinitiation sites described in FIG. 1. It is probable that the −13 startsite is very rare and was not detected by RNase mapping.

Analysis of the sequence of the flanking region (FIG. 1) revealedseveral consensus DNA binding elements: an Sp1 site (−146), a cAMPresponsive element binding (CREB) site (−287; Sassone-Corsi, 1988), anuclear receptor response element (−344 to −356; Parker, 1993), a GATA-3site (−1073; Ko and Engel, 1993), and a weakly degenerate Octamer motif(−522). Moreover, an E-box (−118) contained in a dyad symmetricalelement could be recognized. The proximal region (−245 to +82) also hasan unusually high GC content (67%) and a high number of dinucleotide CpGthat may have some regulatory significance (Antequera and Bird, 1993).Finally, a 20 bp sequence identical to the NRSE (Neural RestrictiveSilencer Element; Mori et al., 1992) or RE1 (Restrictive Element; Kraneret al., 1992) sequence was found in the 3′ end of the 1.2 kbp fragment(+18 to +38).

A 1.2 kbp Fragment of Flanking Sequence of the β2-Subunit Gene PromotesNeuron-Specific Expression In Vitro.

A construct was generated containing the 1163 bp EcoRI/Eco47III fragment(from −1125 to +38) of the β2-subunit 5′ flanking region fused to theLuciferase gene (de Wet et al., 1987) (plasmid EE1.2-Luci). Thepolyadenylation sites of SV40 were inserted upstream from the β2-subunitsequences to avoid readthrough. The transcriptional activity of theplasmid EE1.2-Luci was then tested by transient transfection intopheochromocytoma (PC12) cells, neuroblastoma cell lines NIE 115 andSK-N-Be, SVLT, a striatal cell line (Evrard et al., 1990), NIH3T6 orHeLa fibroblasts and human kidney cell line 293. Using RT-PCR, weverified that the neuroblastomas and the PC12 cells normally express theP2-subunit mRNA but not the striatal SVLT cell lines or the 3T6fibroblasts. FIG. 3 shows that in PC12 cells and neuroblastomas, the 1.2kbp fragment is 20 to 180-fold more active in mediating transcription ofthe reporter gene than in the other cell lines. In fibroblasts, 293cells and SVLT cells, the transcriptional activity of the 1.2 kbpfragment is not significantly higher than that of the promoterlessvector (FIG. 3). Therefore, the β2-subunit promoter is not active inthese cell lines. These in vitro transfection experiments demonstratethat the 1163 bp fragment mimics the expression pattern of theendogenous β2-subunit gene, and thus contains a cell-specific promoter.

The 1163 bp Promoter in Transgenic Mice.

To test the 1163 bp promoter in vivo, the EcoRI/Eco47III fragment waslinked upstream from the nls-β-galactosidase reporter gene (Kalderon etal., 1984). The polyadenylation signals from SV40 were ligateddownstream of the coding sequences. The resulting 4.7 kb fragment wassubsequently microinjected into the male pronuclei of fertilized eggsfrom F1 hybrid mice (C57Bl6xSJL). DNA extracted from the tails of theoffspring was analyzed for the presence of the β-galactosidase gene bythe polymerase chain reaction (PCR). Three independent founders wereobtained and analyzed for expression.

Two lines (13 and 26) had expression in neurons and the third line didnot express at all. This shows that the 1163 bp promoter containsregulatory elements sufficient to drive neuron-specific expression invivo. In the peripheral nervous system PNS, both lines expressed in thesame structure. In contrast, in the CNS the labelling pattern of line 26is a subset of that of line 13. We will only describe line 13 in detail.As expected, most peripheral β2-expressing ganglia expressedβ-galactosidase (β-gal), whereas in the CNS only a subset of β2-positiveregions expressed the β-gal. For instance, FIG. 4C shows that the vastmajority of the neurons of the lumbo-sacral spinal cord express theβ2-subunit transcripts, whereas only a subset of neurons in the ventraland dorsal horns display β-gal activity.

The expression of the transgene could be detected in the peripheralganglia in E10.5 and E11 embryos. The labelling was examined in E13total embryos (FIG. 4A) and in brains at later ages (E17, PO andadulthood). At E13, labelling was prominent in PNS: strong labelling wasobserved in the dorsal root ganglia (DRG, FIGS. 4 and 5 C, D); someganglia associated with the cranial nerves (the trigeminal see FIG. 5A,geniculate, glossopharyngeal and vagal ganglia); the ganglia of thesympathetic chain (FIGS. 5C, D); the ganglionic cells of the retina(FIG. 5A); and putative parasympathetic ganglia in the cardiac wall(FIG. 5B). At E13, clusters of positive cells were also present atseveral levels of the neuraxis, in both the brainstem and theproencephalon. Clusters of stained neurons were also observed in theventral and lateral spinal cord.

Later in development (E17), positive neurons were found clustered inseveral basal telencephalic nuclei whereas dispersed cells were stainedin the caudate-putamen. At the diencephalic level, positive clusterswere present in the zona incerta and reticular thalamic nucleus, and inmany hypothalamic nuclei. In the brainstem, most motor nuclei of cranialnerves (with the exception of the dorsal motor nucleus of the vagusnerve) showed some to high labelling. In addition, the dispersed cellsof the V mesencephalic nucleus appeared strongly stained, as well as thepontine nuclei, the prepositus hypoglossal nucleus and a few dispersedcells in the pontine tegmentum.

At PO in line 13, the distribution of positive cells already appearedmore restricted than at previous ages (for example labelling in basaltelencephalon and oculomotor nuclei was clearly diminished). In the CNSof adult animals labelled cells were detected only in the hypothalamus.In line 13, some clusters of cells were stained in the mucosa of thegastrointestinal tract (stomach and duodenum) and in the pancreas.Ectopic labelling was detected in the genital tubercle and in severalsuperficial muscles of line 13, but none of these tissues were stainedin the line 26.

Identification of a Minimal Cell Specific Promoter

To investigate in more detail the regulatory elements involved in thepromoter activity, we generated a series of plasmids containing 5′deletions of the 1163 bp promoter. These plasmids were tested bytransient transfection into fibroblasts and SK-N-Be cells. These twocell lines were chosen as they were the most easily transfected celllines. Moreover, the neuroblastoma line was initially isolated fromperipheral structures (Biedler et al., 1978) and is a convenient tool tostudy the regulatory elements carried by the 1163 bp promoter.

When 157 bp were deleted from the 5′ end of the 1163 bp promoter(plasmid 1006E-Luci, described in FIG. 1), the luciferase activity didnot significantly change in neuroblastomas but increased in fibroblasts(FIG. 6). When 301 bp were further deleted, the activity of theremaining promoter continued to increase in the fibroblasts but not inneuroblastomas (see plasmid 862E-Luci, FIG. 6). Thus, the 157 and 301 bpdeleted plasmids carry repressor elements which are only active infibroblasts. However, the truncated 862 bp promoter still displayed aneuron-specific activity (FIG. 6, compare activity of 862E-Luci in bothcell lines), showing that additional regulatory elements are carried bythe 1.2 kbp promoter. Moreover, a repressor could be present between−824 and −245 (compare the activities of 862E and 283E-Luci in theneuroblastomas). This putative regulatory element was not furtheranalyzed. Indeed, a 283 bp promoter (plasmid 283E-Luci) is still ˜160times more active in neuroblastomas than in fibroblasts, confirming thepresence of another neuron-specific regulatory elements in this proximalportion of the promoter.

When 150 bp were deleted from the 5′ end of the proximal 283 bppromoter, a very strong decrease of the transcriptional activity wasdetected in both fibroblasts and neuroblastomas (see activity of plasmid133E-Luci). This shows that crucial positive regulatory elements havebeen deleted. These positive and negative elements were furtherinvestigated by deletion and mutation studies of the proximal portion ofthe promoter.

Negative and Positive Regulatory Elements in the Proximal Region.

The 3′ end of the β2-subunit promoter contains putative protein factorbinding sites. To analyze the role of these elements in β2-subunit generegulation, we generated plasmids containing mutations in these bindingsites. Using deletion experiments, an activator was detected between −95and −245 (see FIG. 3, the difference between 283E and 133E-Luci). As theE-box located at nt-118 was a good candidate, we analyzed the effect ofmutations in this element on transcriptional activity. Table 1 shows a40% reduction of the transcriptional activity of the mutated promotercompared to that of the wild type promoter. The role of the E-box innon-neuronal tissues was more difficult to assess as the basal level oftranscription was already low in fibroblasts.

To further understand the role of the E-Box in the regulation of thepromoter, we investigated the protein complexes able to interact withthis sequence. Gel shift assays were performed using the 33 bp sequence(nt-135 to -103, oligonucleotide E-D) as a probe. When the ³²-P labelledoligonucleotide was mixed with nuclear extracts from neuroblastomas orfibroblasts, three complexes were observed (FIG. 7). All of them werefully displaced by an excess of the unlabelled oligonucleotide E-D. Incontrast, no competition was observed when the competitoroligonucleotide was mutated within the E-Box/Dyad (oligonucleotideMut.E, see FIG. 7 lane “Mut-E”). This shows that the E-box/Dyad is theonly element contained within the −135/103 sequence able to bind nuclearprotein. This sequence is likely to be involved in the activity of theβ-subunit promoter.

An NRSE/RE1 sequence is also present in the proximal region and has beenshown to act as a silencer in fibroblasts but not in PC12 cells orneuroblastomas (Kraner et al., 1992; Li et al., 1993; Mori et al.,1992). Point mutation of this sequence in the context of the 1163 bppromoter resulted in a 105-fold increase in the transcriptional activityin fibroblasts, and only a 3-fold increase in neuroblastomas (Table1[[A]]). This sequence is thus responsible for at least part of thecell-specific expression of the β2 subunit gene.

TABLE 1 Positive and negative regulatory elements in the proximal regionof the 1163 bp promoter. Effect of mutations in the proximal part of the1163 bp promoter. The activities of the wild type or mutated promotersare normalized to the luciferase activity of the promoterless KS-Luciplasmid. The activites of EE1.2-Luci are from FIG. 3. NeuroblastomasFibroblasts (3T6) (SK-N-Be) EE1.2-Luci wild type 1.1 (100%) 157 (100%)EE1.2-Luci/NRSE/RE1 115.5 ± 13.8 (1050%) 502 ± 204 (320%)EE1.2-Luci/E-Box ND 94 ± 14 (60%) 

Alignment of the proximal silencer of the β-subunit promoter with otherneuronal promoters:

Mouse β2: (SEQ ID NO: 23) TGCGCGGC.TTCAGCACCACGGACAGCGC.TCCCGTCC SodiumChannel (nt 29): (SEQ ID NO: 24) ATTGGGTT.TTCAGAACCACGGACAGCAC.CAGAGTCTSCG10 (nt 621): (SEQ ID NO: 25) AAAGCCAT.TTCAGCACCACGGAGAGTGC.CTCTGCTTSynapsin I (nt 2070): (SEQ ID NO: 26)CTGCCAGC.TTCAGCACCGCGGACAGTGC.CTTCGCCC CAML1 (nt 1535): (SEQ ID NO: 27)TACAGGCC.TCCAGCACCACGGACAGCAG.ACCGTGAA Calbindin (nt 1093): (SEQ ID NO:28) CCGAACGG.AGCAGCACCGCGGACAGCGC.CCCGCCGC Neurofilament (nt 383): (SEQID NOs 29 and 30, respectively)ATCGGGGT.TTCAGCACCACGGACAGCTC.CCGCGGGGTTCAGCACCACG GACAGCGC

The sequences are taken from (Maue et al., 1990, Na channel, accessionnumber M31433), (Mori et al., 1990; SCG10, M90489), (Sauerwald et al.,1990; Synapsin I, M55301), (Kohl et al., 1992; CAML1 gene, X63509),(Gill and Christakos, 1993, Calbindin gene, L11891), (Zopf et al., 1990;Neurofilament gene, X17102, reverse orientation). The numbering refersto the sequences in the GenBank/EMBL library.

Elimination of High Affinity Nicotine Receptor in Transgenic MiceResults in Alteration of Avoidance Learning

The β2-subunit of the nAChR was disrupted in embryonic stem (ES) cells,and mice deficient in this subunit were subsequently generated (FIG. 8).β2−/−mice were viable, mated normally and showed no obvious physicaldeficits. Overall brain size and organization were normal (see forexample FIG. 9, A and B). Western blot analysis of total brainhomogenates using anti-β2 monoclonal 270¹¹ (FIG. 8 d) andimmunocytochemistry throughout the brain using a polyclonal anti-β2antibody⁹ demonstrated that the immunoreactivity detected in controlmice was absent in β2−/− mice and was diminished in β+/−mice.β2-encoding mRNA was undetectable in β2−/− mice by in situ hybridizationusing β2-antisense oligonucleotides (FIG. 9A).

The distributions of the α4- and β2-subunits largely overlap in thebrain, and these subunits are thought to combine to form the predominantnAChR isoform in the CNS¹². Based on oocyte expression experiments⁶,β4-is the only subunit identified thus far that might also be able toform functional heteropentamers with the α4-subunit. The β4-subunit wasexpressed normally in the brains of β2+/−mice or β2−/− mice, withexpression in the medial habenula (MHb) and the interpeduncular nucleus(IPN)¹⁰, and no upregulation elsewhere in the brain to replace theβ2-subunit (FIG. 9A). Nor was the expression of the α4- (FIG. 9A), α5-or β3-subunit mRNAs significantly altered in mutant mice.

Equilibrium binding experiments have shown that nicotine binds to apopulation of high affinity sites (KD near 10 nM^(13,14)) whosedistribution tallies well with that of the α4- and β2-subunits¹³⁻¹⁵.Quantitative receptor autoradiography was performed using ³H-nicotine (4nM) to visualize high affinity nAChR in brain sections from β2++, +/−and −/− mice (FIG. 9B). Nicotine binding in situ was completelyabolished in β2−/− animals, and was reduced by approximately 50% in allbrain areas in β2+/− animals implicating the β2-subunit in mediatingthis high affinity binding. Electrophysiology of Transgenic Mice.

Neurons of the anterior thalamus, which express very high levels of β2(and α4) subunit mRNAs (FIG. 9A), were studied for anelectrophysiological response to nicotine. This area, easily accessiblein a slice preparation, responded consistently to 10 μM nicotine in wildtype animals with an average inward current of 155+/−73 pA which wasblocked by 1 μM dihydro-β-erythroidine. The agonist order of theresponse was compatible with that seen for α4/β2-containing nicotinicreceptors in vitro⁶ (nicotine>DMPP>cytisine) (FIG. 10A). Anteriorthalamic neurons required several minutes to an hour for completerecovery of the agonist response, suggesting that receptor response isprone to desensitization. Moreover, a relatively high dose of 1 μM wasrequired for a reproducible response, implying that nicotine does notbind to its high affinity site to activate. High affinity nicotinebinding sites may therefore be nAChRs in a desensitized conformation.

In β2−/− mice the response of anterior thalamic neurons to nicotine wascompletely abolished in 100% of neurons tested (FIG. 10B). As a control,neurons in the MHb, where both α3 and β4 are strongly expressed, werealso tested. Nicotine caused an average inward current of 505+/−132 pAin wild type mice, and the agonist potency of this response followed therank order for the α3/β4 containing receptor (cytisine=nicotine>DMPP)(FIG. 10A). As expected, the response of cells in the MHb to nicotinewas maintained in mutant mice.

The β2 subunit is expressed in the ganglia of wild type animals⁸⁻¹⁰, butthere was no apparent difference in heart rate or basal bodytemperature. Spontaneous locomotor activity, which is sensitive to highdoses of nicotine and is not modified by drugs selective for the β2/α4isoform of the nAChR¹⁶, was not significantly different in β2−/−, B+/1and β+/+ mice.

Cognitive and Behavioral Results.

Learning and memory were examined in mutant and wild type mice using twoprocedures. The Morris water maze^(17,18) evaluates spatial orientationlearning. The performance of mutant mice on this test did not differfrom that of wild type mice when tested on the visible platform task, oron the hidden platform task (minimum swim-time reached after 5 days oftraining: mutants (n=8): 7.4+/−1.4 sec; wild type (n=8): 8.2+/−2.0 sec).In the transfer test both groups of animals spent approximately 35% ofthe time in the platform quadrant, with the same number of platformcrossings (mutants: 4+/−0.4; wild type: 3.9+/−0.6).

Retention of an inhibitory avoidance response was assessed using thepassive avoidance test, which was also chosen for its pharmacologicalsensitivity to nicotine administration^(19,20). This test consisted of atraining trial in which the mouse was placed in a well-lighted chamberof a shuttle box, and the latency to enter the adjacent dark chamber wasmeasured. Upon entry to the dark chamber, a mild, inescapable foot shockwas delivered, and vehicle or nicotine (10 μg/kg) was injected into themouse. Twenty-four (24) hours later, retention was assessed by measuringthe latency to enter the dark chamber. Time spent in the light chamber(retention latency) increased proportionally to the applied foot shockin both mutant and wild type mice. However, treatment with nicotineconsistently facilitated retention (p<0.01) by shifting the curve upwardby approximately 80 sec only in wild type mice (FIG. 10A). Nicotineadministration was completely ineffective in mutant mice. Interestingly,retention latency was significantly higher for mutant mice than fortheir non-mutant, vehicle-injected siblings (p<0.05) (FIG. 10B).

Increased retention in the passive avoidance test can be observed inanimals with a decreased pain threshold or increased emotionality.Therefore, further behavioral testing was performed on all mice includedin this experiment. Mutant mice did not differ from their non-mutantsiblings for flinch, vocalization or jump response to foot shock.Emotionality was tested by measuring exploratory activity in a twocompartment apparatus for 15 min^(21,22). The average time spent in thedark compartment, the locomotor activity in the dark compartment and thetransitions between compartments did not differ between the mutant andwild type mice. Therefore, neither changes in pain sensitivity norchanges in emotionality can account for the difference in retentionlatency observed in passive avoidance testing.

Studies using low doses of nicotine²³ or specific nicotinic agonists¹⁶suggest that high affinity nAChRs in the brain mediate the effects ofnicotine on passive avoidance. Accordingly, nicotine cannot change theperformance of β2−/− mice on this test, as they lack high affinitybinding sites. The enhanced performance of mutant mice versus wild typemice is quite surprising, however. Several explanations for theparadoxical effect of the β2-subunit mutation can be proposed. Onehypothesis is that nicotine injection improves performance of wild typemice on passive avoidance as a result of desensitization, and thusinactivation of aAChRs, leading to enhanced performance on the test.Therefore, the behavior of mice lacking the receptor might mimic that ofmice whose receptors have been desensitized²⁴. Another possibility isthat nAChRs may be present in at least two pathways that interact withopposite effects to generate the behavior measured in passive avoidance.If one pathway is physiologically more active than the other, theinactive pathway will be preferentially stimulated by injection ofnicotine in wild type animals, while the more active pathway will bepreferentially influenced by β2-gene inactivation.

The experiments described above demonstrate that nAChRs containing theα-subunit mediate the effects of nicotine on passive avoidance, aspecific learning task. These mice provide a model system for studyingthe pharmacological effects of nicotine in the CNS, and are useful inelucidating the role of high affinity nAChRs in cognitive processes,nicotine addiction, and dementias involving deficits of the nicotinicsystem.

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1-19. (canceled)
 20. A method for isolating the genomic DNA clone forthe β2-subunit of the mouse neuronal nicotinic acetylcholine receptorcomprising providing a mouse genomic DNA library and hybridizing undersuitable conditions a DNA probe encoding the β2-subunit of neuronalnicotinic acetylcholine receptor from another mammalian species. 21-23.(canceled)
 24. A nucleic acid probe consisting essentially of thesequence set forth in FIG. 1 (SEQ ID NO. 22), a sequence hybridizing tothe sequence set forth in FIG. 1 under stringent conditions, or afragment thereof. 25-27. (canceled)
 28. A method for isolating neuronsfrom non-human tissue comprising: providing a transgenic, non-humanmammal all of whose germ cells and somatic cells contain a DNAintroduced into the mammal or an ancestor of the mammal at an embryonicstage, wherein the DNA contains a promoter of the β2-subunit of neuronalnicotinic acetylcholine receptor consisting essentially of the sequenceset forth in FIG. 1 (SEQ ID NO. 22), a sequence hybridizing to thesequence set forth in FIG. 1 under stringent conditions, or a fragmentthereof, wherein the DNA is operatively linked to a nucleotide sequenceencoding a protein, polypeptide, or peptide, and wherein the encodedprotein, polypeptide, or peptide is a reporter gene, identifying theneurons which express the reporter gene, and separating the neurons thatexpress the reporter gene from other cells.
 29. A method for targetingthe expression of a desired polypeptide, protein or peptide product toneurons in a non-human, transgenic mammal comprising replacing the genefor the β2-subunit of neuronal nicotinic acetylcholine receptor with theDNA encoding the desired product in the genome of the mammal byhomologous recombination, where said product is encoded by a DNAsequence.
 30. A transgenic, non-human mammal all of whose germ cells andsomatic cells contain DNA introduced into the mammal or an ancestor ofthe mammal at an embryonic stage, wherein the DNA contains a promoter ofthe β2-subunit of neuronal nicotinic acetylcholine receptor consistingessentially of the sequence set forth in FIG. 1 (SEQ ID NO. 22), asequence hybridizing to the sequence set forth in FIG. 1 under stringentconditions, or a fragment thereof, wherein the DNA is operatively linkedto a nucleotide sequence encoding a protein, polypeptide, or peptide,and wherein the DNA has been mutated by point mutation, deletion,insertion or other means whereby the expression of the protein,polypeptide, or peptide has been altered as measured by biochemicalassay or behavioral assay of a transgenic mammal.
 31. A cell lineproduced from the mammal as claimed in claim
 30. 32-34. (canceled) 35.An isolated DNA consisting essentially of the sequence set forth in FIG.1 (SEQ ID NO. 22), a sequence hybridizing to the sequence set forth inFIG. 1 under stringent conditions, or a fragment thereof, wherein theDNA is operatively linked to a nonhomologous nucleotide sequenceencoding a protein, polypeptide, or peptide, wherein the DNA comprisessequences sufficient to promote transcription of the operatively linkednucleotide sequence in neuronal cells. 36-39. (canceled)
 40. A nucleicacid probe consisting essentially of the sequence encoding at least oneexon of the β2-subunit of neuronal nicotinic acetylcholine receptor. 41.A method of directing expression of a peptide in a cell, comprisingincorporating into the cell a DNA operatively linked to a nucleotidesequence encoding a peptide, polypeptide, or protein, wherein the DNA isselected from the group consisting of: (A) the sequence from aboutnucleotide −1125 to about nucleotide +38 as set forth in FIG. 1 (SEQ IDNO. 22); (B) a sequence having promoter activity, which hybridizes toDNA complementary to said sequence (A) under stringent conditions,wherein said stringent conditions comprise a temperature of about 65° C.and an SSC buffer concentration of about 0.1×SSC; (C) the sequence fromabout nucleotide −968 to about nucleotide +38 as set forth in FIG. 1;(D) the sequence from about nucleotide −824 to about nucleotide +38 asset forth in FIG. 1; (E) the sequence from about nucleotide −245 toabout nucleotide +38 as set forth in FIG. 1; and expressing thepolypeptide.
 42. The method of claim 41, wherein the cell is a neuronalcell.
 43. The method of claim 41, wherein the peptide, polypeptide, orprotein is encoded by a reporter gene.
 44. The method of claim 43,wherein the reporter gene encodes β-galactosidase or Luciferase.
 45. Themethod of claim 41, wherein the peptide, polypeptide, or protein is atoxin, growth factor, neuropeptide, tumorigenic protein, oncogenicprotein, or immortalizing protein.
 46. A method of identifying a cellexpressing a peptide, comprising incorporating into the cell a DNAoperatively linked to a nucleotide sequence encoding a peptide, whereinthe DNA is selected from the group consisting of: (A) the sequence fromabout nucleotide −1125 to about nucleotide +38 as set forth in FIG. 1(SEQ ID NO. 22); (B) a sequence having promoter activity, whichhybridizes to DNA complementary to said sequence (A) under stringentconditions, wherein said stringent conditions comprise a temperature ofabout 65° C. and an SSC buffer concentration of about 0.1×SSC; (C) thesequence from about nucleotide −968 to about nucleotide +38 as set forthin FIG. 1; (D) the sequence from about nucleotide −824 to aboutnucleotide +38 as set forth in FIG. 1; (E) the sequence from aboutnucleotide −245 to about nucleotide +38 as set forth in FIG. 1;expressing the polypeptide; and identifying the cell expressing thepolypeptide.
 47. The method of claim 46, wherein the cell is a neuronalcell.
 48. The method of claim 46, wherein the peptide is encoded by areporter gene.
 49. The method of claim 48, wherein the reporter geneencodes β-galactosidase or Luciferase.
 50. The method of claim 46,wherein the peptide is a toxin, growth factor, neuropeptide, tumorigenicprotein, oncogenic protein, or immortalizing protein.
 51. A method ofisolating a cell expressing a peptide, comprising incorporating into thecell a DNA operatively linked to a nucleotide sequence encoding apeptide, wherein the DNA is selected from the group consisting of: (A)the sequence from about nucleotide −1125 to about nucleotide +38 as setforth in FIG. 1 (SEQ ID NO. 22); (B) a sequence having promoteractivity, which hybridizes to DNA complementary to said sequence (A)under stringent conditions, wherein said stringent conditions comprise atemperature of about 65° C. and an SSC buffer concentration of about0.1×SSC; (C) the sequence from about nucleotide −968 to about nucleotide+38 as set forth in FIG. 1; (D) the sequence from about nucleotide −824to about nucleotide +38 as set forth in FIG. 1; (E) the sequence fromabout nucleotide −245 to about nucleotide +38 as set forth in FIG. 1;expressing the polypeptide; and isolating the cell expressing thepolypeptide.
 52. The method of claim 51, wherein the cell is a neuronalcell.
 53. The method of claim 51, wherein the peptide is encoded by areporter gene.
 54. The method of claim 52, wherein the reporter geneencodes β-galactosidase or Luciferase.
 55. The method of claim 50,wherein the peptide is encoded by a toxin, growth factor, neuropeptide,tumorigenic protein, oncogenic protein, or immortalizing protein.